Cellulose is an abundant, renewable biopolymer that can serve as building blocks for biofuels, bioplastics and other biochemicals. Enzymatic hydrolysis of cellulose to glucose by cellulase is the first step in converting this abundant biomass into bio-based products. Cellulase typically refers to a mixture of enzymes that includes (1) endoglucanases, which attack randomly within the cellulose fiber; (2) exoglucanases, which attack the end of fibers; and (3) beta-glucosidases, which hydrolyze small cellulose fragments to glucose.
Cellulases are typically produced on an industrial scale using Trichoderma reesei, a filamentous fungus that secretes high levels of cellulases when grown aerobically in the presence of cellulase inducers. Cellobiose and lactose are two effective inducers widely used in the industry for cellulase production in Trichoderma reesei. See Kubicek et al., Biotechnology for Biofuels, 2:19 (2009). Due to increasing costs of cellobiose and lactose, however, enzyme manufacturers have turned to alternative inducers.
Sophorose (2-O-beta-D-glucopyranosyl-alpha-D-glucose) has also been recognized as a good inducer for cellulase synthesis in Trichoderma. See Kubicek et al., Biotechnology for Biofuels, 2:19 (2009). Sophorose has been found to be a powerful inducer of a cellulase gene promoter sequence in Trichoderma viride, increasing cellulase production by about 2500 times compared to cellobiose. See Mandels et al., J. Bacteriol., 83(2): 400-408 (1962). The costs associated with sophorose, however, have limited its use in industrial cellulase production.
First, sophorose is considered a “non-gratuitous” inducer because sophorose is metabolized by Trichoderma during fermentation. As a result, sophorose needs to be continually supplied during the fermentation process in order to achieve optimum induction. Second, sophorose is typically produced through glucose reversion reactions using either acids or enzymes. See e.g., England et al., U.S. Published Patent Application No. 2010/0009408; Thompson et al., J. Am. Chem. Soc., 76(5):1309-1311, (1962). These reversion reactions often require reaction times ranging from hours to days. Further, these reversion reactions typically yield a mixture of products with a low concentration of sophorose in a high-concentration glucose background, where typical glucose to sophorose ratios range from 30:1 to 60:1. The sophorose is typically difficult to concentrate and purify cost-effectively from the product mixtures of these reversion reactions.
Other sophorose-containing molecules (also known as sophorosides) have been considered as potential inducers for industrial cellulase production. For example, sophorolipids naturally produced by the yeast Candida bombicola have been found to have protein inducing abilities. See Gross et al., U.S. 2008/0076165; Ju et al, U.S. 2008/0241885. The sophorolipids naturally secreted by Candida bombicola typically consist of one sophorose molecule (about 50% by weight) linked to a hydroxylated fatty acid. Two natural sophorolipids produced by Candida bombicola are depicted in FIG. 1A-B. These natural sophorolipids are usually found as a mixture of lactonic sophorolipids and sophorolipids with fatty acid chains. The compound in FIG. 1A is a naturally produced lactonic sophorolipid containing acetyl groups at both the C6″ and C6′ positions. The compound in FIG. 1B is a naturally produced acidic sophorolipid containing acetyl groups at both the C6″ and C6′ positions. See also Gross et al., U.S. 2008/0076165; Van Bogaert et al., Process Biochemistry, 46(4): 821-833 (2011). Natural sophorolipids, however, have not been observed to have sufficient protein induction ability in Trichoderma to produce cellulases on an industrial scale.
Thus, there exists a need in the art for an inducer for cellulase production, as well as production of other proteins, that can be produced viably on commercial scale.